Download Flow Cytometry

Introduction into
Flow Cytometry
Stephanie Gurka, Andreas Hutloff, Timo Lischke,
Kroczek-Lab, Robert Koch Institute, Berlin
Flow Cytometry - FCM
FACS = Fluorescence Activated Cell Sorting
(also used for analytical cytometers)
• Analysis of the physical properties of single cells
or other biological particles
• Basic principle:
a single cell passes through a flow cell
and is illuminated by a laser source
detection and analysis of scattered / emitted light
• Use of fluorescence marked monoclonal antibodies
multi-parameter analysis (up to 18) for each individual cell
• High flow-rate (> 20,000 cells/sec)
Particles can be measured with a flow cytometer
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•
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Blood cells
Tissue cells
Algae
Protozoa
Chromosomes
Yeast
Prerequisite:
single cell suspension
(Disaggregation: mechanical or enzymatic)
Instrument Overview
Sample
(single cell suspension)
The Laser System
typically used monochromatic laser sources (nm)
• Gas laser systems which require complex air or water cooling
are more and more substituted with diode and solid-state lasers
• Modern Flow-cytometers (e.g. BD LSR II) accomodate up to five lasers
Multi-Laser Systems
time delay
488 nm Laser
633 nm Laser
55 ms
17 ms
405 nm Laser
Optics - Forward Scatter Channel (FSC)
Coherent lightsource
(488 nm)
detect the amount of light scattered in the forward direction
(along the same axis that the laser light is traveling)
voltage
voltage
voltage
Forward Scatter Detector
time
particle passes through the focus
-> scattered light is detected by a photodetector,
-> an electrical pulse is generated and presented to the signal processing electronics.
Intensity of forward scatter
is most influenced by the size of cells
10
FSC-Histogram
9
Cell count
8
7
6
5
4
3
2
1
0
Intensity
FSC-Threshold
• instrument is triggered when the signal exceeds a predefined threshold level
-> reject non-particle events such as debris/noise from optical / electronic sources.
FSC tends to be more sensitive to surface properties of particles (e.g. cell ruffling)
-> can be used to distinguish live from dead cells
Optics - Side Scatter Channel (SSC)
• detect the amount of light scattered to the side
(90° to the axis that the laser light is traveling)
• Intensity of SSC is most influenced by the shape and optical homogeneity of cells
Scatter Plot
SSC tends to be more sensitive to inclusions within cells
-> can be used to distinguish granulated cells from non-granulated cells
Which parameters can be measured?
• the relative size
(FSC)
• the relative granularity
or complexity (SSC)
• the fluorescence intensity (FL1/2,
up to
FL X)
-> Analysis of complex primary samples (heterogeneous cells), such as immune cells
Detection of rare cell types
Using Fluorescence in Flow Cytometry
or cells
transfected with fluorescent proteins
•
•
•
Nucleic acid fluorochromes
Fluorochromes for membrane potential analysis or for ion flux (e.g. Ca2+)
Membrane label fluorochromes
What is Fluorescent Light ?
488 nm
530nm
FITC
Stokes Shift
Incident Light
Emitted Fluorescent Light
The fluorochrome absobs energy from the laser and releases the absorbed energy by:
a) Vibration and heat dissipation
b) Emission of photons of a longer wavelength
=====>
FLUORESCENCE
Stokes shift: energy difference between the wavelength of absorption and emission
Properties of Fluorochromes
Excitation/Emission Spectra
Fluorescence detection
• Fluorescence emitted by each fluorochrome is usually
detected in a unique fluorescence channel.
Fluorescence detector
(PMT3, PMT4 etc.)
SSC detector
photo multiplier tube (PMT)
FCS detector
photo diode
Laser
• Specificity controlled by
the wavelength selectivity of optical filters and mirrors.
signal levels are high
Optical Layout - BD FACSCalibur
Resolution Sensitivity:
Clone RPA-T4
D
W1
W2
Stain Index = D / W
Reagent
Filter
Stain Index
PE
585/40
356.3
Alexa 647
660/20
313.1
APC
660/20
279.2
PE-Cy7
780/60
278.5
PE-Cy5
695/40
222.1
PE-Alexa 610
610/20
80.4
Alexa 488
530/30
75.4
FITC
530/30
68.9
APC-Cy7
7801/60
42.2
Alexa 700
720/45
39.9
Pacific Blue
440/40
22.5
D = difference between positive and negative peak medians
W = 2 x rSD (robust standard deviation)
Fluorescence One Color Histogram
in conjunction with
fluorescence-based protein reporters
(GFP)
-> monitor both transfection efficiency
and protein expression levels.
Data Analyis
cell count
Histogram:
Light/Fluorescence
Intensity
Intensities of 2 Light/Fluorescence Parameters plotted against each other
CD8
2D plot:
CD4
Gating - Statistics
MFI =10
Mean Fluorescence Intensity (MFI)
Quadrant Statistic
% Y+X-
% Y+X+
MFI =150
%A
Y
%C
%B
% Y-X-
% Y-X+
X
Gating - example
Gating - example
Basis of multicolor flow cytometry
A laser beam of a single wavelength
is used to excite several fluorochromes
with different Stokes shifts and, thereby,
produce a variety of fluorescent colors.
Fluorescence dyes used for flow cytometry
http://www.bdbiosciences.com/spectra/
Fluorochrome
excitation
wavelength (nm)
emission
maximum (nm)
LP Mirror
(Canto2)
BP Filter
(Canto2)
FITC, CFSE
488
525
502
530/30
PE
488
575
556
585/42
PI
488
620
PerCP / PerCP-Cy5.5 / PE-Cy5.5
488
675 / 695 / 695
655
670LP
PE‑Cy7
488
767
735
780/60
AF647 / Cy5 / APC
633
665 / 667 / 660
685
660/20
A700
633
723
710
730/45
APC-Cy7
633
767
735
780/60
Pacific Blue / DAPI
405
451 / 460
-
450/50
Pacific Orange / DAPI
405
551 / 460
502
510/50
Knowing the excitation and emission properties of fluorescent compounds:
 Select combinations of fluorochromes that will work together optimally
on a specific flow cytometer with specific lasers !
Fluorescence dyes used for flow cytometry
Fluorochrome
Company
FITC, PE, PerCP, APC, Cy5
Becton Dickinson
Alexa Fluor ___
Molecular Probes
(Invitrogen)
eFluor ___
eBioscience
V ___
BD Horizon
Pacific ___
___
Oregon ___
___
…
…
Quenching
Relative intensity
Fluoresence intensity
FITC pH dependence
Concentration fluorescein
pH
Wanted properties of fluorochromes
Brightness
(high quantum yield)
Photostability
(no bleaching)
pH insensitivity
(stability of fluorescence emission)
Water solubility
(little hydrophobic interactions)
% inital fluoresence intensity
Fluorescence: points to consider
Photobleaching
Photostability / Photobleaching
Instrument compatibility (fit to excitation wavelenght)
Multiparameter
Time (sec)
(small emission spectrum)
Molecular Probes
Types of Fluorochromes
I)
Small dyes:
FITC, Cy5, AlexaFluor´s, eFluor´s,
FITC
I)
I)
g
Large Protein dyes:
phycoerythrin, allophycocyanin, peridinin-chlorophyll-protein
PEB
accessory photosynthetic pigment of red (R-PE, PerCP) or bluegreen algae (APC).
PE:
APC:
PerCP:
240-kDa protein with 34 phycoerythrobilin fluorochromes per molecule.
105-kDa protein with 6 phycocyanobilin chromophores per molecule.
35-kDa protein with phycoerythrobilin fluorochromes
Coupling of fluorescent dyes to antibodies
I) Small dyes / haptens (FITC, Cy5, AFs, Dig, …)
Reaction with primary amine groups of the mAb
fluorescein-5-EX succinimidyl ester
Coupling of fluorescent dyes to antibodies
II) Protein dyes (phycoerythrin, allophycocyanin)
Amine - thiol crosslinking
1) The bifunctional crosslinker Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1carboxylate (SMCC) reacts with amine groups of the fluorescent protein (R1)
thereby introducing a maleimide group
2) The mAb (R2) is partially reduced (with DTT) which yields free sulfhydryl groups
Antibody titration basics
maximize signal:noise (pos/neg separation)
– This may occur at less than saturated staining
– This may or may not be the manufacturer’s recommended titer
increasing amount of mAb
•
10000
signal
noise
S:N
1000
100
10
Intensity
•
1
1000
Titer is affected by:
– Staining volume
– Number of cells
– Staining time and temperature
– Type of sample (whole blood, PBMC, etc.)
100
10
ng antibody
1
Fluorescence Tandem
Basis of multicolor flow cytometry
A laser beam of a single wavelength is used to excite several fluorochromes with
different Stokes shifts and, thereby, produce a variety of fluorescent colors.
Two Color Experiment - 1 Laser
positive population negative population
Filters collect 2 colors
Fluorescence Compensation
positive population
negative population
mathematical subtraction
of the fluorescence due to one fluorochrome
from the fluorescence due to another
PE-MFI (neg) = PE-MFI (pos)
Fluorescence Compensation
Fluorescence Compensation
Compensation controls
• Cells stained with a single fluorochrome-conjugated Ab (analyzed individually)
-> One control for each of the fluorochromes used in the experiment
+ Single control for every tandem conjugate
• Negative and positive populations are required (>10%)
• Use bright markers to setup proper compensation
Small errors
in compensation of a dim control
can result
in large compensation errors
with bright reagents
• CompBeads
• manually (up to 4 FL) or automatic compensation (>4 FL)
Specificity Controls
Controls must undergo the same treatment (i.e., preparation, fixation)
as all the tubes in an experiment.
unstained / control: to detect "auto-fluorescence" or background staining
(monocytes/macrophages, cultured cells, or activated cells)
(to set up PMT-voltage for FSC, SSC and FL-channels)
secondary control: for indirect staining (Bio/SAv, Dig/anti-Dig) - secondary Ab alone
to control for non-specific binding of this polyclonal Ab
to dead or sticky cells.
specificity (experimental and gating) controls:
e.g.
Transfected cells: transfected / mock transfected / wt cell line,
Primary cells:
WT / KO
or
activated / naive
Further Specificity Controls
 not necessary for (lineage) markers with clearly separated populations
Isotype Control:
Ab with the same Ig isotype as the test Ab,
specificity known to be irrelevant to the analyzed sample
-> whether observed fluorescence is NOT due to non-specific
(Fc receptors, dead cells) binding of the fluorescent Ab.
(one for each class of antibody used for staining,
with the same concentration and F/P ratio as Ab of interest)
FMO Control:
Fluorescence Minus One
leaving out the antibody of interest in the staining panel
-> fluorescence spillover of all other fluorochromes in channel of interest.
„Cold Block“:
Preincubation with an excess of unlabeled mAb
prior to addition of fluorophore labeled mAb (no wash between)
All events (cells) with fluorescence above the threshold set with the above controls
are considered positive for the marker of interest.
Comparison of gating controls
Doublet discrimiation
based on fluorescence height, fluorescence area and signal width.
Autofluorescence
• fluorescent signals generated by the cells themselves (from pyridine and flavin nucleotides)
• Present in all cells (viable and dead).
• Adds to fluorescence label of cells -> decreases fluorescence detection limit
• observed in all fluorescence channels,
but decreases dramatically at longer wavelengths (>600 nm, far-red/infra-red).
-> for cell types with high autofluorescence,
a dye with a longer emission wavelength (APC, APC-Cy7)
often provides excellent signal-to-noise ratio.
Dead cell exclusion
Dead cells, with compromised membrane integrity, tend to be sticky
-> bind all sorts of reagents unspecifically. -> exclude dead cells from analysis
• dye exclusion methods with DNA intercalating fluorochromes:
propidium iodide (PI), 7-amino-actinomycin D (7-AAD) or
DAPI staining to positively identify dead cells
by their membrane permeability
• fixable live/dead stain with fluorescent dye
General principle: Dye reacts with free amines.
Live cells (left) react with the fluorescent reactive dye only
on their surface (weakly fluorescent cells).
Cells with compromised membranes (dead, right) react with
the dye throughout their volume (brightly stained cells).
In both cases, the excess reactive dye is washed away.
Signal Separation: different fluorochromes
important for multicolor analysis: choice of which antibody to use with which fluorochrome
(often many "correct" combinations possible)
consider: For any given mAb clone, the signal-to-noise ratio (positive/negative)
can differ depending on the fluorochrome and instrument used
Isotype Control
13.68
75.29
26.84
28.20
FITC
PE
PE-Cy7
APC-Cy7
a- hu CD4 conjugates
Specificity / Non-Specificity: Fc-Receptors
Ab bind to many cell types by their non-specific (Fc) ends.
Monocytes, BC and DC,
professionally bind many Ab through their Fc-receptors.
• Fab or F(ab’)2 fragments
• Blocking of Fc receptors
with polyclonal Ig or specific mAb against Fc-Receptors (species specific!)
-> significantly reduces background staining, (usually not necessary with cell lines)
caution with indirect staining protocols and anti-rat-Ig
(use purified mouse-gamma globulin or mouse serum instead)
Analytical Variables to consider
Sample preparation time, temperature, buffer (pH, salt concentration)
Lysis, digestion, fixation, permeabilisation, washing steps
instrument
number and type of Lasers, Filters, Fluorescence Detectors
->
antibody
fluorochromes/ -combinations
clone, affinity, monoclonal vs. polyclonal, Ig-Isotype, type of Fluorochrome,
concentration, F/P ratio, (may differ from lot to lot)
staining procedure:
Cell number and concentration: depending on the number of events to be analyzed
(due to cell loss during staining approx. 2 times more cells for staining than for analysis)
Cell concentration during staining:
Maximum density for staining is 5x107 cells/ml -> 50 μl staining volume for up to 2.5 Mio cells
20- 30 min at 4°C in the presence of NaN3 to be sure of minimizing capping / internalization/
miscellaneous loss of surface-bound antibodies
Rapid and scalable:
Performed in 96-well plates and in parallel
Multicolor analysis: Choice of Ab-Fluorochrome
1) Choose brightest set of fluorochromes for particular instrument configuration.
1) Choose fluorochromes to minimize the potential for spectral overlap.
- high Compensation for adjacent channels, (FITC vs PE)
- usually low Cross-beam compensation (blue vs red laser)
Exceptions: GFP and very bright FITC signals like CFSE
(also excited by 405 nm  detected in PacO channel);
PE-Cy5.5 / PerCP-Cy5.5 (excited by 633 nm  detected in AF700 channel)
3) Reserve the brightest fluorochromes for “dim” antibodies, and vice versa.
- Highly expressed Antigens will be resolved with almost any fluorophore
- Antigens expressed at lower density might require brighter flurophores
to separate the positive cells adequately from the unlabeled cells
PacO < APC-Cy7 = PacB = FITC = AF700 = PerCP < PE-Cy7 < AF647 = PE = APC
1) Avoid spillover from bright cell populations
into detectors requiring high sensitivity for those populations.
- Strongly expressed Antigens impair the sensitivity/signal resolution of the adjacent channel
- Preferentially, use this channel for Antigens which are not on the same cell as the Ag of interest
5) Take steps to avoid tandem dye degradation, and consider its impact upon results.
Analytical Variables to consider
Data Acquisition, Analysis and Interpretation
Instrument setup and performance
•
adjust and optimize PMT settings (optimal sensitivity)
Set voltages:
Decrease voltages for any detectors where events are off-scale
Increase voltages for any detectors where low-end resolution is poor
• Run single-stained compensation controls for each experiment and set compensation
• Run samples
• Run appropriate controls:
Instrument setup controls (e.g., CompBeads)
Gating controls (e.g., FMO)
Biological controls (e.g., unstimulated samples, healthy donors)
Speed of analysis (high flow rate -> less intensity resolution)
Analytical Variables to consider
Data Analysis / Interpretation
appropriate number of acquired events to ensure reliable results
gating strategy,
• Visually inspect compensation
Create a template
containing dot plots of each color combination of the experiment,
then examine a fully stained sample for possible compensation problems
• Check gating across all samples in the experiment.
Gates may need to be adjusted across donors and/or experimental runs.
-> Avoid classification errors and false conclusions
due to improper compensation and/or gating, or sample artifacts
Ask for interpreting the data, experiment and instrument setup
-> save time and labor
Rare Event Detection
sensitivity and throughput rates enable detection of extremely rare populations and
events (frequencies < 10-6),
■ Hematopoietic stem cells
■ Dendritic cells
■ Residual disease detection (tumor cell enumeration)
■ Antigen-specific T cells
■ Transient transfectants
Dump channels
use of an"dump channel"
significantly improves detection of rare cells or resolution of dim stains (e.g. CD11c).
staining for an antigen not expressed by the cells of interest ("lineage negative„)
-> exclusion of these cells for analysis
e.g.
B220 for murine T cells,
CD3 + CD8 + Ly-6G/C + CD11b for B cells,
CD3 + CD19 for dendritic cells.
-> also exclude cells binding antibodies unspecifically.
Preferentially,
the CasY or A700 channels are used as dump.